The present invention relates to fluorescence-based assays for measuring protein kinase and phosphatase activity.
Phosphorylation and dephosphorylation of proteins, catalysed respectively by protein kinases and protein phosphatases, are key intracellular processes that regulate cellular functions in eukaryotic cells. The reversible phosphorylation of serine, threonine and tyrosine residues in proteins is a highly effective means for regulating the biological properties of proteins so as to modulate such diverse processes as metabolism, cell division, transcription and translation of genes, and signal transduction mechanisms in cells. For reviews, see Hunter, T., Cell, (1995), 80, 225–236; Karin, M., Curr. Opin. Cell Biol., (1991), 3, 467–473. Thus, protein phosphorylation and dephosphorylation are significant events in an organism's maintenance, adaptation, and susceptibility to disease. Dysfunction in protein phosphorylation and dephosphorylation processes can have serious consequences for cellular regulatory mechanisms and for this reason, protein kinases and phosphatases are suitable targets for the development of high throughput screening assays of importance in the development of new therapeutic drug treatments.
Conventional assays for the detection and measurement of kinase activity include those based on radioactive detection methods using [32P]- or [33P]- labelled ATP as a phosphate source for incorporation of phosphate into a target substrate. Methods employing radioactive isotopes typically rely on a separation step to isolate the labelled product, prior to counting in a scintillation counter (Songyang, Z. et al, Nature, (1995), 373, 536–539).
Alternatively, assays involving non-radioactive detection have been employed, for example utilising antibodies to detect phosphorylated proteins and peptides. Detection modalities include fluorescence polarisation and time resolved fluorescence resonance energy transfer.
WO 99/29894 (Epps et al) relates to high throughput screening assays for protein kinases and phosphatases employing fluorescence detection. The methods utilize a competitive immunoassay procedure to determine the amount of substrate that is phosphorylated (or dephosphorylated) by the enzyme. Determination of enzyme activity is achieved by measuring fluorescence polarisation of a labelled antibody-product conjugate. Alternatively, fluorescence quenching or fluorescence correlation spectroscopy may be used.
WO 00/75167 (Sportsman et al) relates to methods for detecting the addition or removal of a phosphate group to or from a substrate by contacting a luminescent peptide with a binding partner that specifically binds to a phosphorylated peptide.
An alternative fluorescent assay method for protein kinase C (PKC) without antibody selection has been reported by Il et al (Analytical Biochemistry, (1991), 195, 148–152). In this assay, an acrylodan-labelled 25-amino acid synthetic peptide incorporating a PKC phosphorylation site is reported to undergo a 20% fluorescence decrease upon phosphorylation. The assay allows detection of PKC to a level of 0.2 nM, while similar concentrations of cyclic AMP-dependent or type II calmodulin dependent protein kinases produced no change in peptide fluorescence. JP 2001–19700-A discloses the use of a fluorogenic substrate for detecting cAMP dependent protein kinase A activity or protein dephosphorylation activity by measuring changes in fluorescence intensity.
In those assays that employ time resolved fluorescence resonance energy transfer (TR-FRET), more than one detection reagent is required. The preparation and addition of such reagents often requires considerable time, effort and expense. Many of the assays are not truly homogeneous in that they require addition of reagents after the reaction has been initiated. The dependence of TR-FRET on the distance between the detection reagents means that substrates must be engineered to meet that need. Furthermore, many of the assays will require multiple labels. It is not possible in most cases to use the natural substrate, even if desired, because the need to biotinylate proteins and add antibodies precludes their use.